Microfluidic devices, systems and methods for performing integrated reactions and separations

ABSTRACT

Microfluidic devices for performing integrated reaction and separation operations. The devices comprise a planar substrate having a first surface with an integrated channel network disposed therein. The reaction region in the integrated microscale channel network has a mixture of at least first and second reactants located therein, wherein the mixture interacts to produce one or more products. The reaction region is configured to maintain contact between the first and second reactants contained within it. The device also includes a separation region in the integrated channel network, where the separation region is configured to separate the first reactant from the product, when the first reactant and product are flowing through the separation region.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is related to U.S. Ser. No. 60/108,628,filed Nov. 16, 1998 and U.S. Ser. No. 09/093,489 filed Jun. 8, 1998.

BACKGROUND OF THE INVENTION

[0002] In the analysis of biological and chemical systems, a number ofadvantages are realized by the process of miniaturization. For example,by miniaturizing analytical and synthetic processes, one obtainsadvantages in: (1) reagent volumes, where reagents are rare and/orexpensive to produce or purchase; (2) reaction times, where mixing orthermal modulation of reactants is a rate limiting parameter; and (3)integration, allowing one to combine multiple preparative andanalytical/synthetic operations in a single bench-top unit.

[0003] Despite the advantages to be obtained through miniaturizedlaboratory systems, or microfluidic systems, early attempts atdeveloping such systems suffered from a number of problems. Ofparticular note was the inability of early systems to control and directfluid movement through microfluidic channels and chambers in order tomix, react and separate reaction components for analysis. Specifically,many of the early microfluidic systems utilized micromechanical fluiddirection system, e.g., microfabricated pumps, valves and the like,which were expensive to fabricate and required complex control systemsto be properly operated. Many of these systems also suffered from deadvolumes associated with the mechanical elements, which preventedadequate fluid control substantially below the microliter or 100nanoliter range. Pneumatic systems were also developed to move fluidsthrough microfluidic channels, which systems were simpler to operate.Again, however, these systems lacked sufficient controllability to movesmall, precise amounts of fluids.

[0004] Pioneering developments in controlled electrokinetic materialtransport have subsequently allowed for the precise control andmanipulation of extremely small amounts of fluids and other materialswithin interconnected channel structures, without the need formechanical valves and pumps. See Published International PatentApplication No. WO 96/04547, to Ramsey. In brief, by concomitantlycontrolling electric fields in a number of intersecting channels, onecan dictate the direction of flow of materials and/or fluids at anunvalved intersection.

[0005] These advances in material transport and direction withinmicrofluidic channel networks have provided the ability to perform largenumbers of different types of operations within such networks. See,e.g., commonly owned Published International Application No. 98/00231 toParce et al., and Published International Application No.98/00705,describing the use of such systems in performing high-throughputscreening operations.

[0006] Despite the wide-ranging utility and relative simplicity of theseadvances, in some cases, it may be desirable to provide simplersolutions to material transport needs within a microfluidic system. Thepresent invention meets these and other needs.

[0007] In particular, the present invention provides material directionmethods and systems that take advantage of certain flow properties ofthe materials, in conjunction with novel structures, to controllablydirect material flow through an integrated microfluidic channelstructure.

SUMMARY OF THE INVENTION

[0008] In a first aspect, this invention provides a microfluidic devicefor performing integrated reaction and separation operations. The devicecomprises a body structure having an integrated microscale channelnetwork disposed therein. The reaction region within the integratedmicroscale channel network has a mixture of at least first and secondreactants disposed in and flowing through the reaction region, whereinthe mixture interacts to produce one or more products. The reactionregion is configured to maintain contact between the first and secondreactants flowing therethrough. The device also includes a separationregion in the integrated channel network, where the separation region isin fluid communication with the reaction region and is configured toseparate the first reactant from the one or more products flowingtherethrough.

[0009] The invention also provides a device for performing integratedreaction and separation operations. The device comprises a planarsubstrate having a first channel disposed in the substrate containing atleast first and second fluid regions. The first fluid region has anionic concentration higher than an ionic concentration of the secondfluid region, and the first and second fluid regions communicates at afirst fluid interface. Second and third channels are disposed in thesubstrate, the second channel intersects and connects the first andthird channels at intermediate points along a length of the first andthird channels, respectively. The device also includes an electrokineticmaterial transport system for applying a voltage gradient along a lengthof the first channel, but not the second channel whichelectrokinetically moves the first fluid interface past the intermediatepoint of the first channel and forces at least a portion of the firstfluid regions through the second channel into the third channel.

[0010] This invention also provides methods of performing integratedreaction and separation operations which include providing amicrofluidic device comprising a body structure having a reactionchannel and a separation channel disposed therein, the reaction channeland separation channel being in fluid communication. At least first andsecond reactants flow through the reaction channel in a first fluidregion. The first and second reactants interact to form at least a firstproduct within the first fluid region. The step of transporting throughthe first channel is carried out under conditions for maintaining thefirst and second reactants and products substantially within the firstfluid region. At least a portion of the first fluid region is directedto the separation channel, which is configured to separate the productfrom at least one of the first and second reactants. The portion is thentransported along the separation channel to separate the product from atleast the first reactant.

[0011] The invention also provides methods of directing fluid transportin a microscale channel network comprising a microfluidic device havingat least first and second intersecting channels disposed therein, thefirst channel being intersected by the second channel at an intermediatepoint. First and second fluid regions are introduced into the firstchannel, wherein the first and second fluid regions are in communicationat a first fluid interface, and wherein the first fluid region has ahigher conductivity than the second fluid region. An electric field isapplied across a length of the first channel, but not across the secondchannel, to electroosmotically transport the first and second fluidregions through the first channel past the intermediate point, whereby aportion of the first fluid is forced into the second channel.

[0012] The invention also provides methods of transporting materials inan integrated microfluidic channel network comprising a first microscalechannel that is intersected at an intermediate point by a secondchannel. First and second fluid regions are introduced serially into thefirst channel and are in communication at a first fluid interface. Amotive force is applied to the first and second fluid regions to movethe first and second fluid regions past the intermediate point. Thefirst and second fluid regions have different flow rates or inherentvelocities under said motive force. The different inherent velocitiesproduce a pressure differential at the first interface that results in aportion of the first material being injected into the second channel.

[0013] The invention also provides methods of performing integratedreaction and separation operations in a microfluidic system, comprisinga microfluidic device with a body, a reaction channel, and a separationchannel disposed therein. The reaction channel is in fluid communicationwith the separation channel. At least first and second reactants aretransported through the first region. The first and second reactants aremaintained substantially together to allow reactants to interact to format least a first product in the first mixture. The first mixture,including the product, is transported to the second region wherein theproduct is separated from at least one of the reactants.

[0014] The invention also provides methods of performing integratedreaction and separation operations in a microfluidic system, comprisinga microfluidic device having at least first and second channel regionsdisposed therein, the first and second channel regions are connected bya first connecting channel. First reactants are introduced into thefirst channel region, the first reactants being contained within a firstmaterial region having a first ionic concentration. The first region isbounded by second regions having a second ionic concentration, thesecond ionic concentration is lower than the first ionic concentration.The first and second material regions are transported past anintersection of the first channel region and the first connectingchannel, whereby at least a portion of the first material region isdiverted through the connecting channel and into the second channelregion.

[0015] In related aspects, the present invention also providesmicrofluidic devices for analyzing electrokinetic mobility shifts ofanalytes, where the device includes a body structure having a firstmicrofluidic channel portion disposed therein, where the first channelportion has substantially no electrical field applied across its length.A second microfluidic channel portion is also included, but where thesecond channel portion has an electrical field applied across itslength. The second channel portion being fluidly connected to the firstchannel portion. The device also includes a pressure source incommunication with at least one of the first channel portion and thesecond channel portion for moving a material through the first channelportion into the second channel portion.

[0016] Relatedly, the present invention also provides methods ofanalyzing materials using the described devices. In particular, themethods of the invention analyze an effect of a first analyte on asecond analyte. The methods steps include contacting the first analytewith the second analyte in a first microfluidic channel portion havingsubstantially no electric field applied across its length. At least aportion of the first analyte and second analyte is transported to asecond channel portion that is in fluid communication with the firstchannel portion and which has an electric field applied across itslength. A change in the electrokinetic mobility of the second analyte,if any, is measured in the second channel portion, where a change in theelectrokinetic mobility of the second analyte is indicative of an effectof the first analyte on the second analyte.

[0017] Similarly provided are methods of analyzing an electrokineticmobility shift in a first analyte, which methods comprise flowing thefirst analyte through a first microscale channel portion havingsubstantially no electrical field applied across it. The first analyteis then introduced into a second microfluidic channel portion. Anelectric field is then applied across a length of the secondmicrofluidic channel portion but not across the length of the firstmicrofluidic channel portion. Finally, an electrokinetic mobility of thefirst analyte is measured under the electric field applied in the secondchannel portion.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1 schematically illustrates an example of a microfluidicdevice incorporating a layered body structure.

[0019]FIG. 2 schematically illustrates a control system forelectrokinetically moving materials within a microfluidic device.

[0020]FIG. 3 illustrates an example of an embodiment of a microfluidicdevice of the present invention for performing integrated reaction andseparation operations. FIG. 3A illustrates the elements of the deviceitself, while FIGS. 3B-3C illustrate the operation of the device intransporting, reacting and separating reaction components within thedevice of FIG. 3A. FIG. 3D illustrates an alternate configuration forthe device shown in FIG. 3A, and FIG. 3E illustrates a close-up view ofan intersection in a device of the invention which incorporatesconductivity measuring capabilities at the intersection for controllinginjection of reaction mixtures into separation channels.

[0021]FIG. 4 illustrates one alternate embodiment of a microfluidicdevice according to the present invention for performing integratedreaction and separation operations. FIG. 4A illustrates the elements ofthe device itself, while FIG. 4B illustrates the operation of the devicein transporting, reacting and separating reaction components within thedevice of FIG. 4A.

[0022]FIG. 5 is a schematic illustration of the pressure profile acrossfluid regions of differing ionic concentration when being transportedthrough a microscale channel by electrokinetic forces.

[0023]FIG. 6 illustrates an alternate device for performing a containedreaction operation followed by a separation operations in a continuousflow mode. FIG. 6A schematically illustrates the structure of the deviceitself, while FIG. 6B schematically illustrates the operation of thedevice.

[0024]FIG. 7 illustrates a microfluidic device channel layout used inperforming integrated operations where the first portion of theoperation requires containment of reactants while the second portionrequires their separation.

[0025]FIG. 8 illustrates the fluorescence signal of rhodamine B andfluorescein monitored at various locations along the main channel duringthe continuous flow mode operation using the device shown in FIG. 7.

[0026]FIG. 9 illustrates the fluorescence signal of rhodamine B andFluorescein monitored at various locations along the main channel andseparation channel during the injection mode operation using the deviceshown in FIG. 7.

[0027]FIG. 10 schematically illustrates alternate devices for carryingout integrated reaction and separation operations. FIG. 10a illustratesan integrated colinear channel for performing reactions and separationsunder pressure and electrokinetic flow, while FIG. 10b illustrates analternate device which includes a separate but connected channel inwhich electrokinetic separations are carried out.

DETAILED DESCRIPTION OF THE INVENTION

[0028] I. General

[0029] A. Desirability for Integration

[0030] In chemical and biochemical analyses, a number of usefulanalytical operations require processes that include two or moreoperational steps. For example, many operations require that a samplematerial undergo some preparative reaction(s) prior to the ultimateanalytical operation. Alternatively, some analytical operations requiremultiple different process steps in the ultimate analytical operation.As a specific example, a large number of operations require a reactionstep and a separation step, which depending upon the analyticaloperation, may be in either order. Such operations are easily carriedout where one is operating at the bench scale, e.g., utilizing reagentvolumes well in excess of 5 or 10 μl, permitting the use of conventionalfluid handling equipment and technology.

[0031] However, when operating in the microfluidic range, e.g., on thesubmicroliter to nanoliter level, conventional fluid handlingtechnologies fail. Specifically, conventional fluidic systems, e.g.,pipettors, tubing, pumps, valves, injectors, and the like, are incapableof transporting, dispensing and/or measuring reagent volumes in thesubmicroliter, nanoliter or picoliter range. While microfluidictechnology provides potential avenues for addressing many of theseissues, early proposals in microfluidics lacked the specific control tooptimize such systems. For example, a great deal of microfluidictechnology to date has been developed using mechanical fluid andmaterial transport systems, e.g., microfabricated pumps and valves,pneumatic or hydraulic systems, acoustic systems, and the like. Thesetechnologies all suffer from problems of inaccurate fluid control, aswell as excessive volume requirements, e.g., in pump and valve deadvolumes. Failing in this regard, such systems are largely inadequate forperforming multiple integrated operations on microfluidic scale fluid orreagent volumes.

[0032] The present invention, on the other hand, provides microfluidicsystems that have precise fluidic control at the submicroliter,nanoliter and even picoliter range. Such control permits the readyintegration of multiple operations within a single microfluidic device,and more particularly, the integration of a reaction operation and aseparation operation, within a single device. Further, microfluidicsystems of the present invention, that incorporate such control alsooffer advantages of automatability, low cost and high orultra-high-throughput.

[0033] In a particular aspect, the microfluidic devices and systems ofthe invention include microscale or microfluidic channel networks thatcomprise a reaction region and a separation region. These two regionsare connected to allow the controlled movement of material from oneregion to the other. As noted above, this is made simpler by precisecontrol of material transport within the channel network. Inparticularly preferred aspects, material transport is carried out usinga controlled electrokinetic material transport system. In alternatepreferred aspects, combined pressure-based and electrokinetic transportsystems are used.

[0034] As used herein, the term “microfluidic” generally refers to oneor more fluid passages, chambers or conduits which have at least oneinternal cross-sectional dimension, e.g., depth, width, length,diameter, etc., that is less than 500 μm, and typically between about0.1 μm and about 500 μm. In the devices of the present invention, themicroscale channels or chambers preferably have at least onecross-sectional dimension between about 0.1 μm and 200 μm, morepreferably between about 0.1 μm and 100 μm, and often between about 1 μmand 20 μm. Accordingly, the microfluidic devices or systems prepared inaccordance with the present invention typically include at least onemicroscale channel, usually at least two intersecting microscalechannels, and often, three or more intersecting channels disposed withina single body structure. Channel intersections may exist in a number offormats, including cross intersections, “T” intersections, or any numberof other structures whereby two channels are in fluid communication.

[0035] The microfluidic devices of the present invention typicallyemploy a body structure that has the integrated microfluidic channelnetwork disposed therein. In preferred aspects, the body structure ofthe microfluidic devices described herein typically comprises anaggregation of two or more separate layers which when appropriatelymated or joined together, form the microfluidic device of the invention,e.g., containing the channels and/or chambers described herein.Typically, the microfluidic devices described herein will comprise a topportion, a bottom portion, and an interior portion, wherein the interiorportion substantially defines the channels and chambers of the device.

[0036]FIG. 1 illustrates a general example of a two-layer body structure10, for a microfluidic device. In preferred aspects, the bottom portionof the device 12 comprises a solid substrate that is substantiallyplanar in structure, and which has at least one substantially flat uppersurface 14. A variety of substrate materials may be employed as thebottom portion. Typically, because the devices are microfabricated,substrate materials will be selected based upon their compatibility withknown microfabrication techniques, e.g., photolithography, wet chemicaletching, laser ablation, air abrasion techniques, injection molding,embossing, and other techniques. The substrate materials are alsogenerally selected for their compatibility with the full range ofconditions to which the microfluidic devices may be exposed, includingextremes of pH, temperature, salt concentration, and application ofelectric fields. Accordingly, in some preferred aspects, the substratematerial may include materials normally associated with thesemiconductor industry in which such microfabrication techniques areregularly employed, including, e.g., silica based substrates, such asglass, quartz, silicon or polysilicon, as well as other substratematerials, such as gallium arsenide and the like. In the case ofsemiconductive materials, it will often be desirable to provide aninsulating coating or layer, e.g., silicon oxide, over the substratematerial, and particularly in those applications where electric fieldsare to be applied to the device or its contents.

[0037] In additional preferred aspects, the substrate materials willcomprise polymeric materials, e.g., plastics, such aspolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, polystyrene, polymethylpentene, polypropylene,polyethylene, polyvinylidine fluoride, ABS(acrylonitrile-butadiene-styrene copolymer), and the like. Suchpolymeric substrates are readily manufactured using availablemicrofabrication techniques, as described above, or from microfabricatedmasters, using well known molding techniques, such as injection molding,embossing or stamping, or by polymerizing the polymeric precursormaterial within the mold (See U.S. Pat. No. 5,512,131). Such polymericsubstrate materials are preferred for their ease of manufacture, lowcost and disposability, as well as their general inertness to mostextreme reaction conditions. Again, these polymeric materials mayinclude treated surfaces, e.g., derivatized or coated surfaces, toenhance their utility in the microfluidic system, e.g., provide enhancedfluid direction, e.g., as described in U.S. patent application Ser. No.08/843,212, filed Apr. 14, 1997 (Attorney Docket No. 17646-002610), andwhich is incorporated herein by reference in its entirety for allpurposes.

[0038] The channels and/or chambers of the microfluidic devices aretypically fabricated into the upper surface of the bottom substrate orportion 12, as microscale grooves or indentations 16, using the abovedescribed microfabrication techniques. The top portion or substrate 18also comprises a first planar surface 20, and a second surface 22opposite the first planar surface 20. In the microfluidic devicesprepared in accordance with the methods described herein, the topportion also includes a plurality of apertures, holes or ports 24disposed therethrough, e.g., from the first planar surface 20 to thesecond surface 22 opposite the first planar surface.

[0039] The first planar surface 20 of the top substrate 18 is thenmated, e.g., placed into contact with, and bonded to the planar surface14 of the bottom substrate 12, covering and sealing the grooves and/orindentations 16 in the surface of the bottom substrate, to form thechannels and/or chambers (i.e., the interior portion) of the device atthe interface of these two components. The holes 24 in the top portionof the device are oriented such that they are in communication with atleast one of the channels and/or chambers formed in the interior portionof the device from the grooves or indentations in the bottom substrate.In the completed device, these holes function as reservoirs forfacilitating fluid or material introduction into the channels orchambers of the interior portion of the device, as well as providingports at which electrodes may be placed into contact with fluids withinthe device, allowing application of electric fields along the channelsof the device to control and direct fluid transport within the device.

[0040] In many embodiments, the microfluidic devices will include anoptical detection window disposed across one or more channels and/orchambers of the device. Optical detection windows are typicallytransparent such that they are capable of transmitting an optical signalfrom the channel/chamber over which they are disposed. Optical detectionwindows may merely be a region of a transparent cover layer, e.g., wherethe cover layer is glass or quartz, or a transparent polymer material,e.g., PMMA, polycarbonate, etc. Alternatively, where opaque substratesare used in manufacturing the devices, transparent detection windowsfabricated from the above materials may be separately manufactured intothe device.

[0041] These devices may be used in a variety of applications,including, e.g., the performance of high throughput screening assays indrug discovery, immunoassays, diagnostics, genetic analysis, and thelike. As such, the devices described herein, will often include multiplesample introduction ports or reservoirs, for the parallel or serialintroduction and analysis of multiple samples. In preferred aspects,however, these devices are coupled to a sample introduction port, e.g.,a pipettor, which serially introduces multiple samples into the devicefor analysis. Examples of such sample introduction systems are describedin e.g., Published International Patent Application Nos. WO 98/00231 and98/00707, each of which is hereby incorporated by reference in itsentirety for all purposes.

[0042] As described above, the devices and systems of the presentinvention preferably employ electrokinetic transport systems formanipulating fluids and other materials within the microfluidic channelnetworks. As used herein, “electrokinetic material transport systems”include systems which transport and direct materials within aninterconnected channel and/or chamber containing structure, through theapplication of electrical fields to the materials, thereby causingmaterial movement through and among the channel and/or chambers, i.e.,positively charged species will generally be attracted to the negativeelectrode, while negative ions will be attracted to the positiveelectrode.

[0043] Such electrokinetic material transport and direction systemsinclude those systems that rely upon the electrophoretic mobility ofcharged species within the electric field applied to the structure. Suchsystems are more particularly referred to as electrophoretic materialtransport systems. Other electrokinetic material direction and transportsystems rely upon the electroosmotic flow of fluid and material within achannel or chamber structure which results from the application of anelectric field across such structures. In brief, when a fluid is placedinto a channel which has a surface bearing charged functional groups,e.g., hydroxyl groups in etched glass channels or glassmicrocapillaries, those groups can ionize. In the case of hydroxylfunctional groups, this ionization, e.g., at neutral pH, results in therelease of protons from the surface and into the fluid, creating aconcentration of protons at near the fluid/surface interface, or apositively charged sheath surrounding the bulk fluid in the channel.Application of a voltage gradient across the length of the channel, willcause the proton sheath to move in the direction of the voltage drop,i.e., toward the negative electrode. Although described aselectrophoretic or electroosmotic, the material transport systems usedin conjunction with the present invention often rely upon a combinationof electrophoretic and electroosmotic transporting forces to movematerials.

[0044] “Controlled electrokinetic material transport and direction,” asused herein, refers to electrokinetic systems as described above, whichemploy active control of the voltages applied at multiple, i.e., morethan two, electrodes. Rephrased, such controlled electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. Controlled electrokinetic material transport isdescribed in Published PCT Application No. WO 96/04547, to Ramsey, whichis incorporated herein by reference in its entirety for all purposes. Inparticular, the preferred microfluidic devices and systems describedherein, include a body structure which includes at least twointersecting channels or fluid conduits, e.g., interconnected, enclosedchambers, which channels include at least three unintersected termini.The intersection of two channels refers to a point at which two or morechannels are in fluid communication with each other, and encompasses “T”intersections, cross intersections, “wagon wheel” intersections ofmultiple channels, or any other channel geometry where two or morechannels are in such fluid communication. An unintersected terminus of achannel is a point at which a channel terminates not as a result of thatchannel's intersection with another channel, e.g., a “T” intersection.In preferred aspects, the devices will include at least threeintersecting channels having at least four unintersected termini. In abasic cross channel structure, where a single horizontal channel isintersected and crossed by a single vertical channel, controlledelectrokinetic material transport operates to controllably directmaterial flow through the intersection, by providing constraining flowsfrom the other channels at the intersection. For example, assuming onewas desirous of transporting a first material through the horizontalchannel, e.g., from left to right, across the intersection with thevertical channel. Simple electrokinetic material flow of this materialacross the intersection could be accomplished by applying a voltagegradient across the length of the horizontal channel, i.e., applying afirst voltage to the left terminus of this channel, and a second, lowervoltage to the right terminus of this channel, or by allowing the rightterminus to float (applying no voltage). However, this type of materialflow through the intersection would result in a substantial amount ofdiffusion at the intersection, resulting from both the natural diffusiveproperties of the material being transported in the medium used, as wellas convective effects at the intersection.

[0045] In controlled electrokinetic material transport, the materialbeing transported across the intersection is constrained by low levelflow from the side channels, e.g., the top and bottom channels. This isaccomplished by applying a slight voltage gradient along the path ofmaterial flow, e.g., from the top or bottom termini of the verticalchannel, toward the right terminus. The result is a “pinching” of thematerial flow at the intersection, which prevents the diffusion of thematerial into the vertical channel. The pinched volume of material atthe intersection may then be injected into the vertical channel byapplying a voltage gradient across the length of the vertical channel,i.e., from the top terminus to the bottom terminus. In order to avoidany bleeding over of material from the horizontal channel during thisinjection, a low level of flow is directed back into the side channels,resulting in a “pull back” of the material from the intersection.

[0046] In addition to pinched injection schemes, controlledelectrokinetic material transport is readily utilized to create virtualvalves which include no mechanical or moving parts. Specifically, withreference to the cross intersection described above, flow of materialfrom one channel segment to another, e.g., the left arm to the right armof the horizontal channel, can be efficiently regulated, stopped andreinitiated, by a controlled flow from the vertical channel, e.g., fromthe bottom arm to the top arm of the vertical channel. Specifically, inthe ‘off’ mode, the material is transported from the left arm, throughthe intersection and into the top arm by applying a voltage gradientacross the left and top termini. A constraining flow is directed fromthe bottom arm to the top arm by applying a similar voltage gradientalong this path (from the bottom terminus to the top terminus). Meteredamounts of material are then dispensed from the left arm into the rightarm of the horizontal channel by switching the applied voltage gradientfrom left to top, to left to right. The amount of time and the voltagegradient applied dictates the amount of material that will be dispensedin this manner.

[0047] A schematic illustration of a system 30 for carrying outanalytical operations within a microfluidic device using controlledelectrokinetic material transport is illustrated in FIG. 2. As shown,the microfluidic device 10, is connected to an electrical controller 34via a series of electrical leads/electrodes 32. The electrodes aredisposed in the reservoirs that are disposed at the termini of thechannels in the channel network within the device 10. The electricalcontroller typically includes a power supply, as well as appropriatecircuitry for regulation of voltage and/or currents applied to each ofthe electrical leads/electrodes 32 to control material transport, asdescribed above. One example of such a power supply is that described incommonly owned Published International Patent Application No. WO98/00707. The system shown, also includes a computer 36, which includesappropriate software or other programming for instructing the electricalcontroller to apply appropriate voltage/current profiles to the variousreservoirs or channel termini in order to achieve a desired materialmovement within the device, e.g., for a given operation. In addition toinstructing the electrical controller, the computer also receives datafrom the controller relating to the electrical parameters within thedevice, e.g., applied current/voltage, resistance, etc., as well asreceiving data from the detector 38. For example, in typicalapplications, the detector 38 is an optical, e.g., fluorescencedetector, which detects relative fluorescence levels within the deviceand reports the data to the computer 36 for storage and subsequentanalysis. The detector is generally disposed adjacent a detection windowthat is disposed in the device, e.g., a translucent or transparentregion of the device 10. Accordingly, the computer is typicallyprogrammed to instruct the operation of the system, as well as receive,store and analyze the data generated by the system.

[0048] Although described for the purposes of illustration with respectto a four way, cross intersection, these controlled electrokineticmaterial transport systems can be readily adapted for more complexinterconnected channel networks, e.g., arrays of interconnected parallelchannels.

[0049] In alternate aspects, the present invention provides microfluidicdevices, systems and methods of using them, for performing reaction andseparation operations within an integrated microfluidic channel network,that utilize different material direction and transport means in orderto ensure reactants in the reaction channel portion are maintainedtogether, while reactants are allowed to separate within the separationchannel portion.

[0050] As described above, the integrated device typically includes atleast a first channel portion that is configured so as to maintainreactants that are flowing through it, together. In the context of thepresent embodiment, this is typically accomplished by driving the flowof the reactants through the first channel portion using apressure-based flow system. By using pressure-based flow, differentreactants do not suffer from biasing effects of differentialelectrophoretic mobilities, as is true under purely electrokineticmaterial transport systems. In operation, first and second analytes thatare to be kept in contact are flowed along the first channel portion, orreaction channel, where that channel portion has substantially noapplied electric field disposed across it. The absence of an electricfield avoids the electrophoretic biasing problem noted above. A secondmicrofluidic channel portion, in fluid communication with the firstchannel portion is then used to perform the separation operation. Inparticular, at least a portion of the reactants that are flowing throughthe first channel portion are introduced into the second channelportion. The second channel portion has an electric field applied acrossits length, in order to promote the electrophoretic separation ofreactants. Application of an electric field is generally carried out asdescribed herein, e.g., via electrodes disposed in electricalcommunication with the termini of the second channel portion, eitherdirectly, or via connecting channels. Typically, the materials flowingthrough the second channel portion have a net flow in one direction,e.g., toward the detection zone, as a result of one or both ofelectroosmotic flow and/or pressure based flow from the first channel.As a result, even species with electrophoretic mobilities opposite tothe desired direction of flow, e.g., away from the detection zone in thesecond channel portion, will still have a net flow in that direction,and thereby permit their detection.

[0051] In those instances where the interaction of the first and secondanalytes has an electrophoretic mobility altering effect on one or bothof the analytes, e.g., resulting in a product that has anelectrophoretic mobility different from one or more of the originalanalytes, the applied electric field within the second channel portionwill result in a separation of the product from the original analytes.The product is then detected, allowing a quantitative determination ofthe interaction of the analytes.

[0052] An exemplary assay that is carried out according to the methodsof the present invention is a nonfluorogenic phosphatase assay whichemploys a phosphorylated fluorescent substrate that is dephosphorylatedby a phosphatase enzyme to yield a more negatively charged fluorescentproduct. Thus, the action of the phosphatase on the phosphorylatedsubstrate has a mobility altering effect on the dephosphorylatedproduct. In the systems described herein, the assay is carried out byflowing the phosphatase enzyme and fluorescent phosphorylated substratethrough the first channel portion by applying a pressure differentialacross the first channel portion, to force or draw the reactants throughthe channel. Because there is no electric field applied across thelength of the channel, there is nothing to cause the separation of thedephosphorylated product from the phosphorylated substrate. The mixtureof product, substrate and enzyme is then directed into the secondchannel portion which has, or is capable of having an electric fieldapplied across its length. When subjected to the electric field, thedephosphorylated fluorescent product has a substantially differentmobility within the second channel portion than the phosphorylatedfluorescent substrate. As these two fluorescent components arephysically separated, they are therefore, separately detectable. Theproduction of the separately detectable species, e.g., substrate andproduct, is indicative that the enzyme has acted on the substrate.Assuming then that one wanted to screen a variety of materials todetermine whether those materials had an effect on the phosphataseactivity, it would merely require introducing those materials into thefirst reaction channel, one at a time, as a third reactant contactingthe phosphatase enzyme and substrate. One would again measure therelative amount of fluorescent product produced, and compare it to acontrol reaction, e.g., where no effector of that interaction waspresent.

[0053] The reaction mixture is optionally introduced into the secondchannel portion as discrete aliquots or plugs, which are then separatedto yield two separate peaks of the detected label, or as a continuousflow of the reaction mixture which produces a constant label signalwhich is interrupted when an effector of the desired interaction isintroduced. Such continuous flow assay formats are described in greatdetail in Published International Patent Application No. WO98/00231,which is incorporated herein by reference. In brief, variations in themobility of the labeled portion of the reaction mixture in discreteregions, e.g., regions where effectors (inhibitors/enhancers) areintroduced, results in an accumulation or depletion of the labeledproduct either before or after the particular reaction region. This isdue to the change in amount of product within those regions resultingfrom the presence of, e.g., an inhibitor, which is then made detectableby the differential mobility of product and substrate.

[0054] B. Specific Assay Examples

[0055] As noted above, a number of useful analytical operations requireprocesses that include two or more operational steps. For example, anumber of analytical assays require the performance of a reaction stepfollowed by a separation step. This is typically the case where theactivity that is sought to be detected in the assay does not itselfproduce a change in the level of a detectable signal, such as theproduction or depletion of a colored, radioactive or fluorescentspecies, e.g., product or substrate, an alteration in detectablesolution characteristics, e.g.. pH, conductivity, etc. or the like. Insuch cases, it is often necessary to be able to separate reactants fromproducts in order to then distinguish between these components anddetermine their relative quantities.

[0056] Specific examples of analytical operations that do not produce analteration in the level of detectable signal in a mixture of reactantsand products are those assays referred to as “non-fluorogenic” or“non-chromogenic” assays. In particular, for a number of assay types,reagents are available that will produce a colored or fluorescent signalin response to a particular activity. For example, for a number ofenzymes, fluorogenic or chromogenic substrates are commerciallyavailable. In the case of fluorogenic substrates, the substrate can beeither non-fluorescent or have a low level of fluorescence as asubstrate. Alternatively, the substrate may be fluorescent while theproduct is non-fluorescent or detectably less fluorescent than thesubstrate. However, upon reaction with the enzyme of interest, afluorescent product is produced (or the fluorescent substrate isconsumed). By measuring the amount of fluorescence produced or consumed,one can determine the relative activity of the enzyme.

[0057] Other examples of fluorogenic reactants include, e.g., nucleicacid or molecular beacons. These molecular beacons include afluorophore/quencher pair, at different ends of a self-complementarynucleic acid sequence or at different ends of two complementary probes.In its native state, autohybridization of the probe or probes places thefluorophore adjacent to the quencher, thereby quenching the fluorescentsignal. However, under denaturing conditions, or when the beacon ishybridized to a complementary nucleic acid sequence, the fluorophore isseparated from its quencher, and a fluorescent signal is detectable.

[0058] In the case of non-fluorogenic assays, however, reagents oftenare not available that will produce an altered fluorescence followingthe reaction of interest, i.e., there is no change in fluorescentquantum efficiency of the product from the substrate, or between thefree and bound (or complexed) reactants. Thus, while a substrate maybear a detectable label, the products of the action of an enzyme on thatsubstrate will bear the same label and be present in the same mixture,and are therefore not separately detectable without, for example, asubsequent separation step. The same is true, for example, where aligand bears a detectable label, and is contacted with a receptor ofinterest in a mixture. The free ligand bears the same label as theligand/receptor complex, and is therefore generally indistinguishablefrom the bound or complexed ligand/receptor in typical fluorescentintensity detection systems, without at least a subsequent separationstep.

[0059] Despite these difficulties however, many reactions do result inchanges in other properties of the reactants/products. For example, inmany cases, a reaction will produce a change in charge and/or size ofthe reactants and/or products. As noted previously, because reactantsand products of these non-fluorogenic assays cannot be distinguishedfrom each other with respect to fluorescence intensity or spectrum, whenpresent in a mixture of the two, it is generally necessary to separatethem prior to detection.

[0060] As in bench scale operations, it is these changes in reactantcharacteristics that are exploited in separating the reactants andproducts in the microfluidic devices of the present invention.Specifically, the devices and systems of the present invention that areused in performing such non-fluorogenic assays, comprise aninterconnected microfluidic channel structure that includes a reactionregion and a separation region. In particularly preferred aspects, thedevices include a channel portion in which reactants are maintainedtogether, in order to allow the reaction to progress. Following thereaction, the unreacted reactants and the products are moved to aseparation channel or channel portion, where separation of the reactantsand products is carried out, followed by detection of the desiredcomponent, typically the product.

[0061] In addition to non-fluorogenic enzyme assays, a number of otherassays are non-fluorogenic or non-chromogenic. For example, with thepossible exception of assays that utilize a molecular beacon, e.g.,certain nucleic acid binding assays, most binding assays arenon-fluorogenic or non-chromogenic. In particular, the bound orcomplexed components of the assay do not change in the amount orspectrum of fluorescence over that of the free components. Thus, in amixture the bound and free components are typically indistinguishable.Again, such assays typically utilize a separation step to firstseparate, then identify the relative levels of bound and freecomponents. In most cases, such assays are carried out by tethering onemember of the binding pair, e.g., the receptor or ligand, or one strandof complementary nucleic acids. The other binding member that bears afluorescent label is then contacted with the tethered member, and thelabeled material that does not bind is washed away, leaving the boundfluorescent, or otherwise labeled material to be detected. This is oneof the basic principles behind the development of molecular arraytechnologies. See, e.g., U.S. Pat. No., 5,143,854, to Pirrung et al.Alternatively, such assays would require the separation of bound andfree components using, e.g., a chromatographic step.

[0062] The devices and systems of the invention are equally applicableto such binding assays, and utilize the same principles as outlinedabove. In particular, bound complexes often have different charges,sizes or charge:mass ratios from their separate reactant components.These differences are exploited, as described above, to separate thereactants, e.g., unbound labeled ligand and unbound receptors, from theproducts, e.g., complexed labeled ligand and receptor. The separatedcomponents are then separately detected, whereby their relativeconcentrations are determined.

[0063] Although described in terms of reactions that employ two or morereactants followed by separation of reactants and the products, it willbe apparent that the methods and devices of the invention are readilyemployed in separating a product from the reactant in a single reactantreaction, e.g., where product is formed from the single reactant, e.g.,a spontaneous reaction (degradation, association, aggregation, etc.), asa result of a thermal or photo-induced reaction (photolysis etc.).

[0064] Related methods are also described in PCT/US98/11969(WO98/56956), and are incorporated herein by reference.

[0065] C. Devices, Systems and Methods

[0066] Integration of multiple different operations within a singlemicrofluidic device can create a number of difficulties. For example, asnoted above, there are a number of difficulties associated withaccurately transporting microscale fluid volumes within integratedchannel structures. However, even more problems arise where differentoperations to be performed within the microscale channels have markedlydiffering, and even conflicting goals. For example, in a number ofanalytical operations, in the reaction portion of the overall operationit is generally desirable to maintain all of the reactants in contactwith one another, to ensure that the reaction will proceed. For theseparation portion of the operation, however, it is generally necessaryto separate those very same reactants from one another, and/or fromtheir products.

[0067] As used herein, the terms “reactant” and “product” are notintended to denote any specific type of interaction, but are generallyused to refer to an interaction between two or more chemical,biochemical or biological species, which interaction includes, chemical,biochemical, electrical, physical or other types of interactions. Somespecific nonlimiting examples of reactants and their respective productsinclude, e.g., complementary single stranded nucleic acids and theirdouble stranded products, ligands and receptors, and the complexesformed therefrom, enzymes and substrates, and the products producedtherefrom, cells and cell affectors and products of such interactions,e.g., agglutinated cells, secreted cellular products, cells withactivated incorporated reporter systems, etc.

[0068] In its simplest embodiment, the operations carried out using thedevices and systems of the invention are performed by providing a firstchannel into which the various reactants are introduced as a continuousmixture. After the reaction has been allowed to occur, a portion of themixture is then aliquoted into a separate channel region in whichseparation of the reaction components occurs. Separation typicallyinvolves a chromatographic or electrophoretic separation of thesecomponents in the separation channel. The separated components are thendetected at a detection window in the separation channel. Althoughdescribed in terms of mixtures of reactants, it will be readilyappreciated that the present invention is useful in performingintegrated reaction and separation operations where a single reactant isintroduced into the system. For example, photolyzable compounds that arefirst photolyzed, then separated, fall within the scope of “reactants”as defined herein. Similarly, heat labile compounds that dissociate(e.g., double stranded nucleic acids), degrade, or hydrolyze underelevated temperatures also fall within this scope.

[0069]FIG. 3A schematically illustrates a microfluidic device forperforming these integrated operations from a top and end view. FIGS. 3Band 3C illustrate the use of the device of FIG. 3A in an “injectionmode,” e.g., where reaction mixtures are injected into a connectedchannel. As shown in FIG. 3A, the device 300, includes a substrate 302that includes a reaction channel 304 that connects a first reactantsource and a waste reservoir 308. As shown, the first reactant source isshown as an inlet 306 from an external sample accessing capillary 306 a,e.g., an electropipettor (See WO 98/00705). A second reactant reservoir310 is fluidly connected to the reaction channel 304 via channel 312. Athird reactant reservoir 314 is connected to the reaction channel 304via channel 316. Separation channel 318 intersects and crosses thereaction channel 304 at a first intersection 320, and connectsseparation buffer reservoir 322 and waste reservoir 324. In operation,the first reactant is introduced into the reaction channel through theexternal sample accessing capillary 306 a. The second reactant is flowedinto the reaction channel from second reactant reservoir 310 via channel312, whereupon it is mixed with the first reactant. An optional thirdreactant is introduced into reaction channel 304 from reservoir 314 viachannel 316. The reaction mixture is flowed through the reaction channel304 past the first intersection 320 and toward the waste reservoir 308.

[0070] A portion of this reaction mixture at the intersection 320 isthen injected into the separation channel 318, which includes anappropriate buffer, medium or matrix for separating the components ofthe mixture. Typically, the separation medium is selected to permit theelectrophoretic separation of the components of the reaction mixture,e.g., reactants and products. Generally, the separation medium isselected to substantially reduce the relative level of electroosmoticflow of fluid within the separation channel, leaving electrophoresis asthe primary force in moving the materials, and through whichdifferentiation of those materials is achieved. In most cases, it issufficient that the separation medium comprises a buffer that includesan ionic strength that is sufficiently high, such that electrophoreticdifferentiation of species is allowed to occur in the channel, e.g.,before electroosmotic flow transports the material into the wastereservoir. In some cases however, e.g., in the separation of largermacromolecules, electrophoretic differentiation of species is enhancedby the incorporation of a sieving component within the separationmedium, e.g., a polymer matrix component. Examples of separation mediaincorporating such matrices have been widely described for use incapillary electrophoresis applications. See, U.S. Pat. Nos. 5,264,101 toDemorest, and U.S. Pat. No. 5,110,424 to Chin. Typically, sievingmatrices are polymer solutions selected from, e.g., agarose, cellulose,polyacrylamide polymers, e.g., cross-linked or non-crosslinkedpolyacrylamide, polymethylacrylamide, polydimethylacrylamide, and thelike. Useful separation matrices also include other types ofchromatographic media, e.g., ion exchange matrices, hydrophobicinteraction matrices, affinity matrices, gel exclusion matrices, and thelike. Similarly, the types of separations performed in the separationchannel can be varied to include a number of different separation types,e.g., micellar electrokinetic chromatography, isoelectric focusingchromatography, counter-current electrophoresis, and the like. In suchcases, the products and reactants from which they are to be separatedhave different partitioning coefficients (vs. different electrophoreticmobilities) in the separation channel.

[0071] The portion of the reaction mixture that is injected into theseparation channel is then transported along the separation channelallowing the components of the mixture to separate. These components arethen detected at a detection window 326 at a point along the separationchannel.

[0072] While the device and methods described above are useful forperforming integrated reaction and separation operations, the throughputof the method as described, is somewhat limited. In particular, in themethod described, only a single reaction is carried out in the reactionchannel 304 at a time. After the separation of the reaction componentshas been carried out in the separation channel 318, new reactioncomponents are introduced into the reaction channel for additionalassays.

[0073] An alternate aspect of the present invention utilizes the samebasic injection mode concept and device structure as that described withreference to FIG. 3A, and is illustrated in FIGS. 3B and 3C. Thisalternate aspect is designed to be utilized in conjunction withhigh-throughput screening assay methods and systems that utilizecontrolled electrokinetic material transport systems to seriallyintroduce large numbers of compounds into a microfluidic channel inwhich a continuous flow assay is carried out. See, commonly assignedpublished International Application No. 98/00231, which is incorporatedherein by reference in its entirety. In carrying out thesehigh-throughput assays, one or more reactants are continuously flowedinto the reaction channel 304 from reservoirs 310 and 314, as shown byarrows 330, 332 and 334. The compound materials (an additional set ofreactants) are introduced from sampling capillary 306 a, and aregenerally maintained together within discrete plugs 336 of material, toprevent smearing of one compound into the next which might result fromelectrophoretic movement of differently charged materials within thecompound plug. These discrete plugs are then contacted with acontinuously flowing stream of one or more additional reactants, e.g.,enzyme and/or substrate, or members of specific binding pairs.

[0074] Maintaining the cohesiveness of the discrete compound/reactantplugs 336 (referred to as “reaction material plugs”) in these flowingsystems, and thus allowing them to react, is typically accomplished byproviding the compound in a relatively high ionic strength buffer (“highsalt buffer” or “high conductivity buffer”), and spacing the compoundplugs with regions of low ionic strength buffer 338 (“low salt buffer”or “low conductivity buffer”). Because most of the voltage drop occursacross the low conductivity buffer regions rather than the highconductivity reaction material plugs, the material is electroosmoticallyflowed through the system before there can be extensive electrophoreticbiasing of the materials in the compound plug 336. In order tosubsequently separate the reactants and products resulting from theassay, as is often necessary in non-fluorogenic assays, the containinginfluence of the high salt plugs/low salt spacer regions must generallybe overcome or “spoiled.”

[0075] In accordance with the method described above, and with referenceto FIG. 3C the containing influence of the high conductivity materialplug 336/low conductivity spacer region 338, is overcome or spoiled byinjecting a portion 340 of the high conductivity reaction material plug336 into the separation channel 318 that is also filled with a highconductivity buffer, as the plug 336 moves past the intersection of thereaction channel and separation channel. As noted above, because theseparation channel is filled with a high conductivity buffer, theelectrokinetic mobility of materials within the channel resulting fromthe electrophoretic mobility of the components of the reaction materialrelative to the electroosmotic movement of the fluid is accentuated.

[0076] As the reaction material plug is transported past theintersection 320 of the reaction channel 304 and the separation channel318, it is injected into the separation channel 318 by switching theflow through the separation channel, as shown by arrow 342. This isgenerally carried out by first slowing or halting flow of the reactionmaterial plug through the reaction channel 304 while that plug 336traverses the intersection 320. Flow is then directed through theseparation channel to inject the portion of the plug that is in theintersection, into the separation channel 318. Controlling flow streamsare also optionally provided at the intersection 320 during thereaction, injection and separation modes, e.g., pinching flow, pull-backflow, etc., as described above and in published InternationalApplication No. 96/04547, previously incorporated herein by reference.

[0077] While this method is very effective, and is also applicable tohigh throughput systems, there is a measure of complexity associatedwith monitoring the progress of the reaction material plugs through thereaction channel and timing the injection of material into theseparation channel. In one aspect, the passage of reaction materialplugs through the intersection 320 is carried out by measuring theconductivity through the intersection, e.g., between reservoirs 322 and324. In particular, because the reaction materials are contained in highionic concentration plugs, their passage through the intersection willresult in an increase in conductivity through the intersection andthrough the channel between reservoirs 322 and 324. Measurement ofconductivity between reservoirs 322 and 324 is generally carried outusing either a low level of direct current, or using an alternatingcurrent, so as not to disturb the electrokinetic flow of materials inthe integrated channel network. Further, because electrokinetictransport is used, electrodes for measuring the conductivity throughchannels are already in place in the wells or reservoirs of the device.Alternatively, smaller channels are provided which intersect thereaction channel on each side, just upstream of the injection point orintersection, as shown in FIG. 3E. Specifically, channels 352 and 354are provided just upstream of intersection 320, and include electrodes356 and 358 in electrical contact with the unintersected termini ofthese channels. As used herein, the term “electrical contact” isintended to encompass electrodes that are physically in contact with,e.g., the fluid such that electrons pass from the surface of theelectrode into the fluid, as well as electrodes that are capable ofproducing field effects within the medium with which they are inelectrical contact, e.g., electrodes that are in capacitive contact orionic contact with the fluid. These electrodes are then coupled with anappropriate conductivity detector 360 for measuring the conductivity ofthe fluid between the electrodes, e.g., in the reaction channel 304, asit flows into the intersection, which flow is indicated by arrow 362.Conductivity is then measured across these channels to identify when thereaction material plug is approaching the intersection. Thisconductivity measurement is then used to trigger injection of a portionof the reaction material plug into the separation channel 318.Typically, each of these additional channels includes a reservoir at itsterminus distal to the reaction channel, and conductivity is measuredvia electrodes disposed in these reservoirs. Alternatively, the twodetection channels could be provided slightly staggered so that thedistance between the channels along the length of the reaction channelis small enough to be spanned by a single reaction material plug. Theelectrodes disposed at the termini of these channels are then used tosense the voltage difference between the intersection of each of the twochannels and the reaction channel, e.g., along the length of thereaction channel. When a high conductivity reaction material plug spansthe distance between the two channels, the voltage difference will beless, due to the higher conductivity of the fluid between them.

[0078] Another preferred method of addressing this issue is describedwith reference to FIG. 3D. In particular, as shown, the device has asimilar layout to that of the device shown in FIG. 3A. However, in thisaspect, the separation channel portion is channel portion 350, which iscolinear with the reaction channel portion 304, channel portion 318 afunctions as a waste/gating channel, and the detection window 336 a isdisposed over channel portion 350. This method of transporting thematerial from the reaction channel region 304 to the separation channelregion 350 is referred to as a “continuous flow mode” or “gatedinjection mode.”

[0079] In operation, the reaction material plugs are directed along thereaction channel portion 304 through intersection 320, and into wastechannel 318 a, toward reservoir 324, e.g., using an electrokinetic gatedflow. During operation of the device, the resistance level betweenreservoirs 322 and 324 is monitored. As a reaction material plug enterswaste channel 318 a, the increase in conductivity resulting from thehigher ionic concentration of the high salt reaction material plug isused to trigger a gated injection of a portion of that plug into theseparation channel 350. Specifically, upon sensing a predetermined levelof conductivity increase, a computer linked with the electricalcontroller aspect of the overall system, directs a switching of theapplied currents to produce the gated flow profile described above, fora short period, e.g., typically less than 1 second. By gating flow ofthe reaction material plugs into waste channel 318 a, conductivitychanges between reservoirs 322 and 324 are more pronounced as the lengthof the plug occupies a greater percentage of the channel across whichthe conductivity is being measured. As a result, one can moreeffectively identify meaningful conductivity changes and therebydetermine when the reaction material plugs enter theintersection/injection point. Specifically, when using this lattermethod, one is measuring conductivity changes resulting from the lengthof the material plug, as opposed to measuring the changes resulting fromthe width of the plug, e.g., as it passes through an intersection acrosswhich conductivity is measured, as described with reference to FIGS.3B-3C, above. Again, as described with reference to FIG. 3E above,auxiliary channels and reservoirs may be used to measure conductivitychanges across different portions of a channel or intersecting channels,e.g., one conductivity sensing electrode may be placed in contact withthe reaction channel, e.g., via a side channel, upstream of theintersection while another is placed downstream of the intresection.

[0080] Although described in terms of detecting changes in conductivity,a number of methods can be used to detect when the reaction materialplug is present in or near the intersection. For example, markercompounds may be provided within either the reaction material region orthe spacer regions. These compounds, and thus the presence or absence ofa reaction material plug or region then can be detected at or near theinjection intersection to signal a change in the flow profile fromreaction to injection mode, e.g., injecting the reaction material intothe separation channel portion. Such marker compounds optionally includeoptically detectable labels, e.g., fluorescent, chemiluminescent,calorimetric, or colloidal materials. The marker compounds are typicallydetected by virtue of a different detectable group than that used todetect the results of the reaction of interest. For example, where thereaction of interest results in a fluorescent product that must beseparated from a fluorescent reactant prior to detection, the markercompound typically includes either a non-fluorescent compound, e.g.,colored, colloidal etc., or a fluorescent compound that has a excitationand/or emission maximum that is different from the product and/orreactant. In the latter case, the detection system for detecting themarker compound is typically configured to detect the marker compoundwithout interference from the fluorescence of the product/reactantlabel.

[0081] In preferred aspects, these marker compounds are neutral (have nonet charge) at the operating pH of the system, so that they are notelectrophoretically biased during transport within their discreteregions. Except as described above, these optically detectable markercompounds are typically detected using a similar or identical detectionsystem used to detect the separated elements of the reaction ofinterest, e.g., a fluorescent microscope incorporating a PMT orphotodiode, or the like.

[0082]FIG. 4A schematically illustrates an alternative mechanism forovercoming the influence of these high salt plug/low salt spacer regionswithin the separation region or channel of the device using anotherversion of the injection mode. As shown, the device 400, includes asubstrate 402, having a reaction channel 404 disposed therein. As shown,the reaction channel 404 is in communication at one end with the inletfrom a pipettor capillary 406 (shown from a top view). The pipettor 406is capable of accessing and introducing large numbers of differentsample materials into the analysis channel 404. The analysis channel isin communication at the other end, with a waste reservoir 408.Reservoirs 410 and 414 typically include the different reactants neededfor carrying out the reaction operation for the device and are connectedto reaction channel 404 via channels 412 and 416, respectively.Separation channel 418 is located adjacent to analysis channel 404, andconnecting channel 420 links the two channels at an intermediate pointin both channels. Separation channel 418 links separation bufferreservoir 422 and waste reservoir 424. A detection window 426 is alsoprovided within separation channel 418, through which separated samplecomponents may be detected.

[0083] In one mode, the device shown in FIG. 4 is capable of takingadvantage of certain flow characteristics of fluids under electrokinetictransport. In particular, in electrokinetically moving different fluidregions that have different electroosmotic flow rates, pressuregradients are created within the fluid regions. In particular,electroosmotic fluid flow within a microscale channel is driven by theamount of voltage drop across a fluid region. Thus, low ionic strength,e.g., low conductance, high resistivity, fluid regions have higherelectroosmotic (“EO”) flow rates, because these regions drop a largeramount of voltage. In contrast, higher ionic strength fluids, e.g.,higher conductance materials, drop less voltage, and thus have lower EOflow rates.

[0084] Where a system includes different fluid regions having differentionic strengths, these different flow rates result in pressuredifferentials at or near the interface of the two fluid regions.Specifically, where a first fluid of higher ionic strength, e.g., asample material, is being pushed by a second fluid region of lower ionicstrength, the trailing end of the first fluid region is at a higherpressure from the force of the second fluid region. Where the firstfluid region is following the second fluid region, the pulling effect ofthe second fluid region results in a lower pressure region at theleading edge of the first fluid region. A channel that includesalternating high and low ionic strength fluid regions, will also includealternating high and low pressure areas at or near the interfaces of thedifferent regions. FIG. 5 schematically illustrates the pressuregradients existing in a channel having such different ionic strengthregions. These pressure effects were described and a method forovercoming them set fort in commonly owned published InternationalApplication No. WO 98/00705, incorporated herein by reference in itsentirety. In brief, in order to prevent perturbations resulting fromthese pressure effects at channel intersections, the channelintersecting the main channel is typically made shallower, as thepressure effects drop off to the third power with decreasing channeldepth, whereas electroosmotic pumping is only reduced linearly withchannel depth. See Published International Application No. WO 98/00705.

[0085] The operation of the device shown in FIG. 4A is described below,with reference to FIGS. 4A and 4B in the performance of ahigh-throughput screening assay, which screens for affectors of areaction of two reactants, e.g., inhibitors or enhancers of enzymeactivity, inhibitors or enhancers of ligand receptor binding, or anyother specific binding pair. In brief, the reactants are maintained in arelatively low ionic strength buffer, and are placed into the firstreactant reservoir 410, and the second reactant reservoir 414. Each ofthese reactants is then electrokinetically transported through thereaction channel 404 toward waste reservoir 408 in a continuous stream,as indicated by arrows 430, 432 and 434. This electrokinetic transportis carried out, as described above, by applying appropriate voltagegradients between: (1) the first reactant reservoir and the wastereservoir; and (2) the second reactant reservoir and the wastereservoir.

[0086] Periodically, a plug of material 436 that includes a compoundwhich is to be screened for an effect on the reaction of the tworeactants is introduced into the reaction channel by way of the externalsample accessing capillary 406 shown from an end view. The capillary 406is integrated with the reaction channel 404. In particularly preferredaspects, this external sample accessing capillary 406 is anelectropipettor as described in published International PatentApplication No. WO 98/00705.

[0087] As described above, these plugs 436 of compound material are in arelatively high ionic strength buffer solution, and are introduced withspacer regions 438 of relatively low ionic strength buffer. The higherionic strength compound plugs typically approach physiological ionicstrength levels, and are preferably from about 2 to about 200 times theconductivity of the low ionic strength buffer, in some cases, from about2 to about 100 times the conductivity of the low ionic strength buffer,and more preferably, from about 2 to about 50 times the conductivity ofthe low ionic strength buffer, and in many cases from about 2 to about20 or even 10 times the conductivity of the low ionic strength buffer.Typically, the high ionic strength buffer has a conductivity from about2 mS to about 20 mS, while the low ionic strength buffer has aconductivity of from about 0.1 mS to about 5 mS, provided the low ionicstrength buffer has a lower conductivity than the higher ionic strengthbuffer.

[0088] As the plugs of material 436 are transported along the reactionchannel, the two reactants are allowed to react in the presence of thecompound that is to be screened, within the plug 436, and in the absenceof the compound to be screened, outside of the plug 436, e.g., withinspacer region 438. As the reaction material plug 436 moves past theintersection of reaction channel 404 and connecting channel 420, thepressure wave caused by the differential flow rates of the high ionicstrength plugs and low ionic strength spacer regions causes a smallportion of the material plug, or “aliquot,” 440 to be injected into theconnecting channel 420.

[0089] As shown in FIG. 5, the pressure wave caused by the interface ofthe high salt and low salt regions is reciprocated at the oppositeinterface of the next compound plug. As such, it is important totransport the aliquot 440 through the connecting channel 420 into theseparation channel 418 and away from the intersection of these channels,before it is sucked back into the reaction channel 404. This isgenerally accomplished by providing the connecting channel withappropriate dimensions to permit the aliquot to progress entirelythrough the connecting channel and into the separation channel.Typically, the connecting channel will be less than 1 mm in length,preferably less than 0.5 mm in length, more preferably, less than 0.2 mmin length, and generally, less than about half the width of the reactionchannel, e.g., typically from about 5 to about 100 μm. Additionally, toprevent refluxing of the aliquot into the reaction channel, flow istypically maintained within the separation channel to move the aliquot440 away from the intersection of connecting channel 420 and separationchannel 418, which flow is indicated by arrows 442. This same injectionprocess is repeated for each compound plug that is serially introducedinto the reaction channel. The effects of the pressure wave at theintersection, and thus the size of the injected plug can be adjusted byvarying the depth of the connecting channel at the intersection, asdescribed above. For example, smaller injections are achieved by makingthe connecting channel shallower than the reaction channel.

[0090] The separation buffer within separation channel 418 is selectedso as to permit separation of the components within the aliquot ofreaction material. For example, whereas the materials in the reactionchannel are contained in a high salt plug to prevent electrophoresis,the separation channel typically includes a high salt buffer solution,which then allows the electrophoretic separation of the components,e.g., by diluting the low salt regions and their effects on materialmovement in the channels, e.g., increased electroosmotic flow ascompared to the electrophoretic effects on the components of thereaction material. Of course, in some cases, a high salt buffer is usedin order to create a more uniform conductivity throughout the separationchannel, allowing separation of components in the aliquot of reactionmaterial before the material is electroosmotically transported out ofthe separation channel.

[0091] As described, in alternate or additional aspects, the separationchannel includes a separation matrix, or sieving polymer, to assist inthe separation of the components of the reaction material aliquot.

[0092] Once the reaction material is injected into the separationchannel 418 it is transported through the separation channel andseparated into its component elements. Typically, the flow of materialwithin the separation channel is directed by electrokinetic means.Specifically, a voltage gradient is typically applied between separationbuffer reservoir 422 and waste reservoir 424, causing the flow ofmaterial through the separation channel. In addition, the voltagegradient within the separation channel 418, is typically applied at alevel whereby there is no current flow through the connecting channel420, or only sufficient current to prevent leakage through theconnecting channel during non-injection periods. This prevents theformation of any transverse currents between the separation channel andthe reaction channel, which might disturb controlled material flow. Onceseparated, the components of the reaction material are then transportedpast a detection window 426 which has an appropriate detector, e.g., afluorescence scanner, microscope or imaging system, disposed adjacent toit.

[0093] Optionally, the device illustrated in FIG. 4 employs activematerial transport, e.g., electrokinetic transport, to inject a portion440 of the reaction material plug 436 into the separation channel 418.In particular, the reaction material plug 436 is electrokineticallytransported along the reaction channel 404, as described above. Once thereaction material plug 436 reaches the intersection of the reactionchannel 404 and the connecting channel 420, the electrical potentials atthe various reservoirs of the device are switched to cause current flow,and thus, flow of a portion of the reaction material, through theconnecting channel, into the separation channel 418. The portion 440 ofthe reaction material plug is then electrokinetically transportedthrough separation channel 418 by virtue of current flow between thereservoirs 422 and 424. The current through the separation channel isadjusted to match the current flowing through the reaction channel 404,so that no transverse currents are set up through the connectingchannel. This active electrokinetic injection, as well as the morepassive pressure differential injection described above, provideadvantages over other injection modes of integrated reaction anseparation, by permitting the reaction and separation channels tooperate at the same time. Specifically, transport of material along thereaction channel does not need to be arrested during the separationprocess, and vice versa.

[0094] A simpler embodiment of the present invention and particularly amicrofluidic device for carrying it out, is illustrated in FIG. 6. Inthis embodiment, the containing influence of the high salt plugs in thereaction region or channel of the device, as described above, isovercome or spoiled by introducing a stream of separation inducingbuffer into the system at the junction between the reaction andseparation regions. As used herein, the term “separation inducingbuffer” refers to a buffer in which molecular species may be readilyseparated under appropriate conditions. Such buffers can include pHaltering buffers, sieving buffers, varied conductivity buffers, bufferscomprising separation inducing components, e.g., drag enhancing oraltering compounds that bind to the macromolecular species to createdifferential separability, and the like. For example, in the systems ofthe present invention, the separation inducing buffer generally refersto either a high salt or low salt buffer introduced into the system atthe junction point between the reaction and separation regions. Theintroduction of high salt or low salt buffer lessens the conductivitydifference between the reaction material plug (typically in high saltbuffer) and the spacer region (typically in low salt buffer), bydiluting out or spoiling the differential electrophoretic/electroosmoticforces among the different regions. This dilution or spoiling allowselectrophoretic separation of the materials in the plug, as describedabove. This method is referred to as a “continuous flow mode” becausethe reaction material plugs are continuously flowing along a colinearchannel, without being redirected into an intersecting channel.Typically, the separation inducing buffer will be either: (1) a highsalt buffer having a conductivity that is greater than the conductivityof the low salt buffer regions, e.g., from about 2 to about 200 timesgreater, preferably from about 2 to about 100 times greater, morepreferably, from about 2 to about 50 times greater, and still morepreferably, from about 2 to about 20 times greater, and often from about2 to about 10 times greater than the conductivity of the low salt bufferregions; or (2) a low salt buffer having a conductivity that is lowerthan the first conductivity by the same factors described above. Ofcourse, implied in these ranges are separation inducing buffers thathave conductivity that is substantially approximately equivalent toeither of the high salt fluid regions or low salt fluid regions.

[0095] As shown in FIG. 6A, the device 600 is disposed in a planarsubstrate 602, and includes a reaction channel region 604 and aseparation channel region 606. The reaction and separation channels arein communication at a junction point 610. Waste reservoir 608 isdisposed at the terminus of the separation channel region 606. Alsointersecting these channels at the junction point 610, is an additionalchannel 612 which delivers high conductivity buffer from reservoir 614into the separation channel region. As with the device described above,reactants are delivered into the junction point 610 for reaction channelregion 604 and separation channel region 606, from first and secondreactant reservoirs 616 and 618 via channels 620 and 622. Compounds thatare to be screened for effects on the reaction of the reactants aretypically introduced using an appropriate external sample accessingcapillary or pipettor 624, e.g. an electropipettor.

[0096] In operation, the reactants are transported from their respectivereservoirs 616 and 618 and along the reaction channel region 604 in acontinuous flow stream, as indicated by arrows 630, 632 and 634.Periodic plugs of compounds to be screened 636 in high salt buffer arealso flowed along the reaction channel, the reaction mixture of thefirst and second reactants and the test compound being contained withinthe high salt plug 636 and adjacent low salt regions. As the plug ofmaterial 636 is transported past the junction point 610, a stream ofhigher conductivity buffer, indicated by arrow 638, continuously mixeswith the reaction mixture plug and adjacent low ionic strength regionschanging the relative field strengths across the high and low ionicstrength regions, e.g., the voltage drop across the lower ionic strengthregions is decreased. This change in field strengths allowsdifferentially charged material components within the reaction mixtureplug 636 to be separated into their component species 640 and 642, basedupon differences in the electrophoretic mobility of those components, asthey move along the separation channel region 606. It should be notedthat in accordance with the present invention, a lower salt buffer couldalso function as a “spoiling buffer” to bring the relative ionicstrengths of the different material regions closer together, and exposethe entire length of the channel to similar voltage gradients, e.g.,including the components of the reaction mixtures.

[0097] Examples of a device and system for performing integratedreaction/separation operations using a combination of pressure flow andelectrokinetic transport are schematically illustrated in FIGS. 10a and10 b.

[0098] As shown in FIG. 10a, the device 1000 includes a body structure1002 which includes a first channel portion 1004 that is fluidlyconnected to a second channel portion 1006. The first channel portion isalso fluidly connected to sources of reactants 1008 and 1010, viachannels 1012 and 1014, respectively. The first channel portion is alsoshown in fluid connection with an external capillary element (not shown)via port 1016. As shown, the second channel portion 1006 is fluidlyconnected to ports/reservoirs 1018, 1020, and 1022 via channel portions1024, 1026 and 1028, respectively. As shown, the device 1000 alsoincludes a detection window 1030 disposed across the second channelportion 1006.

[0099] In operation, first and second analytes, e.g., enzyme andsubstrate, ligand and receptor, etc., are introduced into the firstchannel portion 1004, from reservoirs 1008 and 1010, via channels 1012and 1014, respectively. The first and second analytes are moved into thefirst channel portion by applying an appropriate pressure differentialbetween the reservoirs and the first channel. In the device shown, thisis optionally accomplished by applying a vacuum to reservoir 1022, whichis translated into the first channel portion 1004 by channels 1028 and1006. A third analyte is introduced into the first channel portion 1004through the capillary element (not shown) via inlet port 1016. Again,the vacuum applied to the system functions to draw material that isplaced into contact with the open end of the capillary element.Specifically, the capillary element is dipped into a source of at leasta third reactant whereby the vacuum sips the reactant into the capillarychannel and into channel portion 1004. The first, second and optionallythird reactants are permitted to react as they move along the firstchannel portion 1004 toward the intersection with channel portion 1006.As no electric field is applied across this channel portion 1004, noelectrophoretic separation of the reactants and/or their products willoccur.

[0100] Once the reaction mixture moves into channel portion 1006, it issubjected to an electric field to promote electrophoretic separation ofthe species therein. The electric field is typically applied acrosschannel portion 1006 by placing electrodes into contact with fluid thatis disposed in reservoirs 1020 and 1018, creating an electric fieldbetween the reservoirs and across channels 1024, 1006, and 1026. As thereaction components separated, the separation is detected at detectionwindow 1030, typically as a fluorescent signal, or deviation from asteady state fluorescent signal.

[0101] An alternate device construction for carrying out the same assaymethods is illustrated in FIG. 10b. Components of the device shown inFIG. 10b that are the same as those shown in FIG. 10a are referencedwith the same reference numerals. As shown, the device 1000 includes afirst channel portion 1004 that is fluidly connected to at least firstand second reactant sources, e.g., reservoirs 1008 and 1010, andincludes the optional inlet port 1016 fluidly coupled to an externalcapillary element (not shown). The first channel portion is fluidlycoupled to a vacuum port/reservoir 1032. An additional channel 1034intersects and crosses the first channel portion 1004 and is fluidlyconnected to reservoir/port 1036.

[0102] As with the device illustrated in FIG. 10a, a second channelportion 1038 is used to perform the separation operation. The separationchannel portion connects reservoirs 1040 and 1042, and is fluidlyconnected to channel portion 1004 via channel 1034.

[0103] In operation, the reaction mixture, as described with referenceto FIG. 10a, is drawn into the first channel portion by applying avacuum to reservoir/port 1032. The reaction mixture then moves acrossthe intersection of channel portion 1004 and channel 1034. A portion ofthe reaction mixture at this intersection is then injected into thesecond channel portion 1038. Injection of the reaction mixture from thefirst channel portion 1004 into the second channel portion is preferablyaccomplished by applying an electrical filed across channel 1034, e.g.,between reservoir/port 1036 and 1042 or 1040. Once a plug of thereaction mixture is introduced into the second channel portion,application of an electric field across the second channel portion 1036,e.g., between reservoirs 1042 and 1040, then causes the electrophoreticseparation of the different reaction components, thereby allowing theirdetection at detection window 1030. One of the advantages this latterchannel structure offers over that shown in FIG. 10a is the ability toinject discrete plugs of reaction mixture into the separation channel.In particular, only a small volume of reaction material is injected intothe second channel portion for separation. However, this adds complexitywhen performing higher throughput assays, which are typically simpler ina continuous flow system, e.g., as shown in FIG. 10a.

[0104] The invention is further described with reference to thefollowing nonlimiting examples.

EXAMPLES

[0105] The following examples demonstrate the efficacy of the methodsand devices of the present invention in performing integratedcontainment or reaction and separation operations. For these examples, amicrofluidic device having the channel geometry shown in FIG. 7 wasused. In these experiments, a low salt buffer containing 50 mM HEPES atpH 7.5, and a high salt buffer containing 50 mM HEPES+100 mM NaCl at pH7.5 were prepared. A second high salt buffer (“ultra high salt buffer”),containing 50 mM HEPES+200 mM NaCl at pH 7.5, was prepared and used asthe “spoiling buffer” in the continuous flow mode. A neutral dye,Rhodamine B, and an anionic dye, Fluorescein, were placed in the highsalt buffer in well 3 of the device shown in FIG. 7, and used as markersto track electrophoretic containment and separation in all experiments,because these dyes have different electrophoretic mobilities.

Example 1

[0106] Continuous Flow Mode Reaction/Separation

[0107] In the continuous flow mode, e.g., as described above withreference to FIG. 7, above, the buffer wells of the device shown in FIG.7 were loaded as follows: low salt buffer was loaded in wells 1 and 4,high salt buffer with dyes was loaded in well 3, high salt buffer wasloaded in well 6, and ultra high salt buffer was loaded in wells 2 and8. The following voltages and currents were applied to the listed wells,to direct movement of the materials through the device using an eightchannel current based electrical controller which included a series ofpin electrodes inserted into the wells: 1 2 3 4 5 6 7 8 Time(s) FlowProfile 500 V 10 μA  0 μA 0.5 μA 0 V 0 μA 0 V 10 μA 20 Fill channelw/low salt 500 V  0 μA  0 μA −7 μA 0 V 10 μA  0 V  0 μA 4 Create guardbands 500 V  0 μA 10 μA −7 μA 0 V 0 μA 0 V  0 μA 1 Inject sample 500 V10 μA  0 μA 0.5 μA 0 V 0 μA 0 V 10 μA 10 Move sample downchannel/separate

[0108] To monitor the degree of containment and separation of dyes, thelocation of the detection point was varied along the channel path of dyeflow, and the plotted signals for each detection point are provided inthe panels of FIG. 8. This series of plots clearly indicate that thedyes are contained in the high-low salt format before the injectionpoint (Panel A). The containment is successfully disrupted, e.g., thecontaining influence is overcome, upon the addition of the spoilingbuffer into the main channel, leading to separation of dyes downstream(Panels B, C, D and E).

Example 2

[0109] Injection Mode

[0110] In the injection/separation flow mode, the wells were loaded asfollows: low salt buffer in wells 1 and 4, high salt buffer with dyes inwell 3, high salt buffer in wells 6, 2, and 8. Controlling currents andvoltages were applied as follows: 1 2 3 4 5 6 7 8 Time(s) Flow Profile500 V 0 μA 0 μA 3 μA 0 V 0 μA 0 V 0 μA 10 Fill channel w/low 500 V 0 μA−.5 μA −7 μA 0 V 10 μA  0 V 0 μA 4 Create guard bands 500 V 0 μA 10 μA−7 μA 0 V 0 μA 0 V 0 μA 2 Inject sample 500 V 0 μA 0 μA 3 μA 0 V 0 μA 0V 0 μA 2.8 Move sample down main channel 0 μA 10 μA  0 μA 0 μA 0 V 0 μA0 V 100 V 0.5 Cross inject sample into second channel 500 V 0 μA 0 μA 3μA 0 V 0 μA 0 V 0 μA 10 Clear main channel −.5 μA 10 μA  0 μA 0 μA 0 V 0μA O V 100 V 60 Move sample down separation channel

[0111] The location of the detection point along the main and separationchannels again was varied to monitor the degree of containment of thetwo dyes. FIG. 9 summarizes the results of the dye signals graphically.Once again, the dyes were clearly contained in the high-low salt formatbefore the injection point, (panels A and B) and were cleanly separatedby electrophoresis in the separation channel (panels C and D).

[0112] In summary, these experimental results demonstrated thefeasibility of both the continuous flow and stop flow approaches forintegrating electrophoretic containment and electrophoretic separationin the same microfluidic device.

[0113] The discussion above is generally applicable to the aspects andembodiments of the invention described in the claims.

[0114] Moreover, modifications can be made to the methods apparatus andsystems described herein without departing from the spirit and scope ofthe invention as claimed, and the invention can be put to a number ofdifferent uses including the following.

[0115] The use of a microfluidic integrated system or device forperforming any of the methods and assays set forth herein, particularlythe use of the devices and integrated systems set forth herein forperforming any of the assays or methods set forth herein.

[0116] The use of any microfluidic system or device as described hereinfor performing integrated reaction and separation operations, mobilityshift operations, or any other operation set forth herein, e.g., foranalysis of one or more analytes, as set forth herein.

[0117] Use of an assay or method utilizing a feature or operationalproperty of any one of the microfluidic systems or devices describedherein, e.g., for practicing any method or assay set forth herein.

[0118] Use of kits comprising any device, device element, or instructionset, e.g., for practicing any method or assay set forth herein, or forfacilitating practice of any method or use of any device or system setforth herein, including maintenance kits for maintaining the devices orsystems herein in an appropriate condition to practice the methods andassays set forth herein. While the foregoing invention has beendescribed in some detail for purposes of clarity and understanding, itwill be clear to one skilled in the art from a reading of thisdisclosure that various changes in form and detail can be made withoutdeparting from the scope of the invention. For example, all thetechniques and apparatus described above may be used in variouscombinations which will be apparent upon complete review of theforegoing disclosure and following claims. All publications and patentapplications listed herein and the references cited within thosedocuments are hereby incorporated herein by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Although thepresent invention has been described in some detail by way ofillustrations and examples for purposes of clarity and understanding, itwill be apparent that certain changes and modifications may be practicedwithin the scope of the appended claims.

What is claimed is:
 1. A microfluidic device for performing integratedreaction and separation operations, comprising: a body structure havingan integrated microscale channel network disposed therein; a reactionregion within the integrated microscale channel network, the reactionregion having a mixture of at least first and second reactants disposedin and flowing through the reaction region, the mixture interacting toproduce one or more products, wherein the reaction region is configuredto maintain contact between the first and second reactants flowingtherethrough; and a separation region in the integrated channel network,the separation region in fluid communication with the reaction regionand being configured to separate the first reactant from the one or moreproducts flowed therethrough.
 2. The microfluidic device of claim 1,wherein the reaction region comprises a microscale reaction channelhaving first and second ends and the separation region comprises amicroscale separation channel having first and second ends.
 3. Themicrofluidic device of claim 2, wherein the reaction channel and theseparation channel are in fluid communication and cross at a firstintersection between the first and second ends of the reaction channeland the separation channel, respectively.
 4. The microfluidic device ofclaim 3, further comprising an electrokinetic material transport systemoperably coupled to the first and second ends of the reaction channeland the first and second ends of the separation channel forelectrokinetically transporting material through the reaction channeland into the separation channel.
 5. The microfluidic device of claim 4,wherein at least two of the first and second reactants and product havedifferent electrophoretic mobilities under an applied electric field. 6.The microfluidic device of claim 4, wherein the reaction channelcomprises first and second fluid regions disposed therein, the firstfluid region comprising the first and second reactants and the product,and having a first conductivity, the first fluid region being bounded bythe second fluid regions, wherein the second fluid regions have a secondconductivity that is lower than the first conductivity.
 7. Themicrofluidic device of claim 3, wherein the separation channel comprisesa separation inducing buffer, the separation inducing buffer having aconductivity that is higher than the second conductivity.
 8. Themicrofluidic device of claim 3, wherein the separation channel comprisesa separation inducing buffer, the separation inducing buffer having aconductivity that is lower than the first conductivity.
 9. Themicrofluidic device of claim 3, wherein the separation channel comprisesa separation inducing buffer, the separation inducing buffer having aconductivity that is the same as the first conductivity.
 10. Themicrofluidic device of claim 3, further comprising: at least first andsecond conductivity measuring electrodes disposed in electrical contactwith opposite sides of the reaction channel adjacent to the firstintersection; and a conductivity detector operably coupled to the firstand second conductivity measuring electrodes.
 11. The microfluidicdevice of claim 3, further comprising at least a third reactant in thereaction region, the second and third reactants interacting to producethe product, and wherein the first reactant comprises a test compound.12. The microfluidic device of claim 3, wherein the separation channelcomprises a separation medium disposed therein.
 13. The microfluidicdevice of claim 3, further comprising: a source of at least firstreactant in fluid communication with the reaction channel; and a sourceof at least second reactant in fluid communication with the reactionchannel.
 14. The microfluidic device of claim 13, wherein the source ofat least first reactant comprises at least a first reactant reservoirconnected to the reaction channel via a first reactant channel, and thesource of at least second reactant comprises: a source of at least asecond reactant separate from the body structure; and an external sampleaccessing capillary in fluid communication with the reaction channel,for contacting the second reactant reservoir and transporting a volumeof the second reactant into the reaction channel.
 15. The microfluidicdevice of claim 13, wherein the source of at least first reactantcomprises a first reactant reservoir disposed in the body structure andconnected to the reaction channel via a first reactant channel, and thesource of second reactant comprises a second reactant reservoir disposedin the body structure and connected to the reaction channel via a secondreactant channel.
 16. The microfluidic device of claim 3, wherein thebody structure comprises at least first and second planar substrates, aplurality of grooves being fabricated into a first planar surface of thefirst substrate, and a first planar surface of the second substratebeing mated to the first planar substrate of the first substratecovering the plurality of grooves and defining the integrated channelnetwork.
 17. The microfluidic device of claim 16, wherein at least oneof the first and second substrates comprise a silica-based substrate.18. The microfluidic device of claim 17, wherein the silica-basedsubstrate is selected from glass, quartz, fused silica, or silicon. 19.The microfluidic device of claim 18, wherein the silica based substratecomprises glass.
 20. The microfluidic device of claim 16, wherein atleast one of the first and second substrates comprises a polymericmaterial.
 21. The microfluidic device of claim 20, wherein the polymericmaterial is selected from polymethylmethacrylate, polycarbonate,polytetrafluoroethylene, polyvinylchloride, polydimethylsiloxane,polysulfone, polystyrene, polymethylpentene, polypropylene,polyethylene, polyvinylidine fluoride, andacrylonitrile-butadiene-styrene copolymer.
 22. The microfluidic deviceof claim 21, wherein the polymeric material comprisespolymethylmethacrylate.
 23. The microfluidic device of claim 3, whereinchannels in the integrated channel network have at least onecross-sectional dimension between about 0.1 and about 500 μm.
 24. Themicrofluidic device of claim 3, wherein channels in the integratedchannel network have at least one cross-sectional dimension betweenabout 1 and about 100 μm.
 25. The microfluidic device of claim 24,wherein channels in the integrated channel network have at least onecross-sectional dimension between about 10 and about 100 μm.
 26. Themicrofluidic device of claim 2, wherein the reaction channel comprisesalternating first and second fluid regions, the first region having ahigher ionic concentration than the second fluid region, the reactionmixture being localized in a first fluid region.
 27. The microfluidicdevice of claim 2, wherein the reaction channel and the separationchannel are in fluid communication via a connecting channel, theconnecting channel intersecting the reaction channel between the firstand second ends of the reaction channel, and intersecting the separationchannel between the first and second ends of the separation channel. 28.The microfluidic device of claim 27, further comprising anelectrokinetic material transport system operably coupled to the firstand second ends of the reaction channel and the first and second ends ofthe separation channel for electrokinetically transporting materialthrough the reaction channel and into the separation channel.
 29. Themicrofluidic device of claim 28, wherein at least two of the first andsecond reactants and product have different electrophoretic mobilitiesunder an applied electric field.
 30. The microfluidic device of claim27, wherein the connecting channel comprises a smaller cross-sectionalarea than the first or second channels.
 31. The microfluidic device ofclaim 27, wherein the connecting channel comprises a length less thanabout 1 mm.
 32. The microfluidic device of claim 27, wherein theconnecting channel comprises a length less than about 0.5 mm.
 33. Themicrofluidic device of claim 32, wherein the reaction channel comprisesfirst and second fluid regions disposed therein, the first fluid regioncomprising the first and second reactants and the product, and having afirst conductivity, the first fluid region being bounded by the secondfluid regions, wherein the second fluid regions have a secondconductivity that is lower than the first conductivity.
 34. Themicrofluidic device of claim 32, wherein the separation channelcomprises a separation inducing buffer, the separation inducing bufferhaving a conductivity that is higher than the second conductivity. 35.The microfluidic device of claim 34, wherein the separation inducingbuffer comprises a conductivity that is from about 2 to about 100 timesgreater than the second conductivity.
 36. The microfluidic device ofclaim 34, wherein the separation inducing buffer has a conductivity thatis lower than the first conductivity.
 37. The microfluidic device ofclaim 34, wherein the separation inducing buffer comprises aconductivity that is from about 2 to about 100 times less than the firstconductivity.
 38. The microfluidic device of claim 34, wherein theseparation inducing buffer has a conductivity approximately equal to thefirst conductivity.
 39. The microfluidic device of claim 27, furthercomprising: at least first and second conductivity measuring electrodesdisposed in electrical or capacitive contact with opposite sides of thereaction channel adjacent to the first intersection; and a conductivitydetector operably coupled to the first and second conductivity measuringelectrodes.
 40. The microfluidic device of claim 27, further comprisingat least a third reactant in the reaction channel, the second and thirdreactants interacting to produce the product, and wherein the firstreactant comprises a test compound.
 41. The microfluidic device of claim27, wherein the separation channel comprises a separation mediumdisposed therein.
 42. The microfluidic device of claim 27, wherein thereaction region comprises alternating first and second fluid regions,the first region having a higher ionic concentration than the secondfluid region, the reaction mixture being localized in a first fluidregion.
 43. The microfluidic device of claim 2, wherein the first end ofthe reaction channel is in fluid communication with the first end of theseparation channel at a first junction, and further comprising a bufferchannel having first and second ends, the first end of the bufferchannel in fluid communication with the reaction channel and theseparation channel at the first junction, the second end of the bufferchannel being in fluid communication with a source of separationinducing buffer.
 44. The microfluidic device of claim 43, wherein thefirst and second channel portions are co-linear.
 45. The microfluidicdevice of claim 43, further comprising an electrokinetic materialtransport system operably coupled to the second ends of the reactionchannel, the separation channel and the buffer channel forelectrokinetically transporting material from the reaction region to theseparation region, and for introducing separation inducing buffer intothe separation channel from the buffer channel.
 46. The microfluidicdevice of claim 45, wherein at least two of the first and secondreactants and product have different electrophoretic mobilities under anapplied electric field.
 47. The microfluidic device of claim 43, whereinthe reaction channel comprises first and second fluid regions disposedtherein, the first fluid region comprising the first and secondreactants and the product, and having a first conductivity, the firstfluid region being bounded by the second fluid regions, wherein thesecond fluid regions have a second conductivity that is lower than thefirst conductivity.
 48. The microfluidic device of claim 43, wherein theseparation inducing buffer has a conductivity that is greater than thesecond conductivity.
 49. The microfluidic device of claim 48, whereinthe separation inducing buffer has a conductivity that is from about 2to about 100 times greater than the second conductivity.
 50. Themicrofluidic device of claim 48, wherein the separation inducing bufferhas a conductivity that is lower than the first conductivity.
 51. Themicrofluidic device of claim 48, wherein the separation inducing bufferhas a conductivity that is from about 2 to about 100 times less than thefirst conductivity.
 52. The microfluidic device of claim 48, wherein theseparation inducing buffer has a conductivity that is approximatelyequal to the first conductivity.
 53. The microfluidic device of claim48, wherein the separation inducing buffer has a conductivity that isapproximately equal to the second conductivity.
 54. The microfluidicdevice of claim 43, further comprising at least a third reactant in thereaction region, the second and third reactants interacting to producethe product, and wherein the first reactant comprises a test compound.55. The microfluidic device of claim 43, wherein the separation channelcomprises a separation medium disposed therein.
 56. The microfluidicdevice of claim 43, wherein the reaction region comprises alternatingfirst and second fluid regions, the first region having a higher ionicconcentration than the second fluid region, the reaction mixture beinglocalized in a first fluid region.
 57. A microfluidic device forperforming integrated reaction and separation operations, comprising: abody structure; a first channel disposed in the body structure, thefirst channel having disposed therein, at least first and second fluidregions, the first fluid region having an ionic concentration higherthan an ionic concentration of the second fluid region, and the firstand second fluid regions communicating at a first fluid interface;second and third channels disposed in the body structure, the secondchannel intersecting and connecting the first and third channels atintermediate points along a length of the first and third channels,respectively; an electrokinetic material transport system for applying avoltage gradient along a length of the first channel, but not the secondchannel, to electrokinetically move the first fluid interface past theintermediate point of the first channel, and force at least a portion ofthe first fluid regions through the second channel into the thirdchannel.
 58. The device of claim 57, wherein the first fluid region hasa conductivity that is from about 2 to about 200 times greater than aconductivity of the second fluid regions.
 59. The device of claim 57,wherein the first fluid region has a conductivity that is from about 2to about 100 times greater than a conductivity of the second fluidregions.
 60. The device of claim 57, wherein the first fluid region hasa conductivity that is from about 2 to about 50 times greater than aconductivity of the second fluid regions.
 61. The device of claim 57,wherein the first fluid region has a conductivity that is from about 2to about 20 times greater than a conductivity of the second fluidregions.
 62. The device of claim 57, wherein the first fluid region hasa conductivity that is from about 2 to about 10 times greater than aconductivity of the second fluid regions.
 63. The device of claim 57,wherein the first fluid region comprises at least first and secondmaterials.
 64. The device of claim 63, wherein the first and secondmaterials have different electrophoretic mobilities under an appliedelectric field.
 65. A method of performing integrated reaction andseparation operations, comprising: providing a microfluidic devicecomprising a body structure having a reaction channel and a separationchannel disposed therein, the reaction channel and separation channelbeing in fluid communication; flowing at least first and secondreactants through the reaction channel in a first fluid region, thefirst and second reactants interacting to form at least a first productwithin the first fluid region, wherein the step of transporting throughthe first channel is carried out under conditions for maintaining thefirst and second reactants and products substantially within the firstfluid region; directing at least a portion of the first fluid region tothe separation channel, the separation channel being configured toseparate the product from at least one of the first and secondreactants; and transporting the portion along the separation channel toseparate the product from at least first reactant.
 66. The method ofclaim 65, wherein: the flowing step comprises applying a first voltagegradient along the reaction channel to electrokinetically move the firstfluid region into the intersection; and the directing step comprisesapplying a second voltage gradient along the separation channel todirect at least a portion of the first fluid region into the separationchannel; the separating step comprises applying a third voltage gradientalong the separation channel to separate the first reactant from thefirst product.
 67. The method of claim 66, wherein the conditionssuitable for maintaining the first and second reactant and productsubstantially within the first fluid region comprises the first fluidregion having a first conductivity and being bounded by second fluidregions having a second conductivity that is lower than the firstconductivity.
 68. The method of claim 66, wherein the first conductivityis from about 2 to about 100 times greater than the second conductivity.69. The method of claim 66, wherein the separation channel has aseparation inducing buffer disposed therein, the separation bufferhaving a conductivity lower than the first conductivity.
 70. The methodof claim 66, wherein the separation channel has a separation inducingbuffer disposed therein, the separation inducing buffer having aconductivity approximately equivalent to the first conductivity.
 71. Themethod of claim 66, wherein the product and at least one of the firstand second reactants have different electrophoretic mobilities under anapplied electric field.
 72. The method of claim 65, further comprisingthe step of detecting the separated product in the separation channel.73. The method of claim 65, wherein: in the providing step, the reactionchannel and the separation channel disposed in the body structure are influid communication and cross at a first intersection; the flowing stepcomprises flowing the first fluid region into the first intersection;and the directing step comprises directing the portion of the firstmixture in the intersection into the separation channel.
 74. The methodof claim 73, further comprising the step of detecting when the firstfluid region is disposed in the intersection.
 75. The method of claim74, wherein the step of detecting when the first fluid region isdisposed in the intersection comprises detecting a change inconductivity of fluid at the intersection.
 76. The method of claim 74,wherein the first and second fluid regions have optical characteristicsthat are distinguishable from each other, and the step of detecting whenthe first fluid region is disposed in the intersection comprisesdetecting within the intersection, the optical characteristicsindicating the presence of the first fluid region.
 77. The method ofclaim 74, wherein the optical characteristics that are distinguishablefrom each other comprise a fluorophore or chromophore disposed within atleast one of the first or second fluid regions.
 78. The method of claim77, wherein the optical characteristics that are distinguishable fromeach other comprise a first chromophore or fluorophore disposed in thefirst fluid region and a second chromophore or fluorophore disposed inthe second fluid region, the first fluorophore or chromophore beingdistinguishable from the second chromophore or fluorophore.
 79. Themethod of claim 69, wherein: in the providing step, the reaction channeland the separation channel are in fluid communication via a connectingchannel the connecting channel intersecting the reaction channel at afirst intersection and intersecting the separation channel at a secondintersection; the flowing step comprises flowing the first fluid regioninto the first intersection; and the directing step comprises directingat least a portion of the first fluid region through the connectingchannel into the separation channel.
 80. The method of claim 79, whereinthe directing step comprises providing a voltage gradient between thereaction channel and separation channel to electrokinetically direct aportion of the first fluid region from the reaction channel, through theconnecting channel and into the separation channel.
 81. The method ofclaim 79, wherein the directing step comprises flowing the first fluidregion along the reaction channel through the first intersection, apressure differential present at an interface of the first and secondfluid regions forcing a portion of the first fluid region into theconnecting channel and into the separation channel.
 82. The method ofclaim 69, wherein: in the providing step, the reaction channel has firstand second ends, the separation channel has first and second ends, thefirst end of the reaction channel being in fluid communication with thefirst end of the separation channel at a first junction, and furthercomprising a buffer channel having first and second ends, the first endof the buffer channel in fluid communication with the reaction channeland separation channel at the first junction; the flowing step comprisesflowing the first fluid region along the reaction channel to the firstjunction; and the directing step comprises directing the portion of thefirst mixture in the intersection into the separation channel.
 83. Themethod of claim 82, wherein the directing step comprises directing atleast a portion of the first fluid region into the separation channelwhile concomitantly injecting the separation inducing buffer from thethird channel into the separation channel.
 84. A method of directingfluid transport in a microscale channel network, comprising: providing amicrofluidic device having at least first and second intersectingchannels disposed therein, the first channel being intersected by thesecond channel at an intermediate point; introducing first and secondfluid regions into the first channel, wherein the first and second fluidregions are in communication at a first fluid interface, and wherein thefirst fluid region has a higher conductivity than the second fluidregion; applying an electric field across a length of the first channel,but not across the second channel, to electroosmotically transport thefirst and second fluid regions through the first channel past theintermediate point, whereby a portion of the first fluid is forced intothe second channel.
 85. A method of transporting materials in anintegrated microfluidic channel network, comprising: providing a firstmicroscale channel that is intersected at an intermediate point, by asecond channel; introducing first and second fluid regions serially intothe first channel, the first and second fluid regions being incommunication at a first fluid interface; applying a motive force to thefirst and second fluid regions to move the first and second fluidregions past the intermediate point, the first and second fluid regionshaving different flow rates under said motive force, the different flowrates producing a pressure differential at the first interface, thepressure differential resulting in a portion of the first material beinginjected into the second channel.
 86. The method of claim 86, whereinthe motive force comprises an electric field applied across a length ofthe first channel.
 87. A method of performing integrated reaction andseparation operations in a microfluidic system, comprising: providing amicrofluidic device comprising a body, and a reaction channel and aseparation channel disposed therein, the reaction channel being in fluidcommunication with the separation channel; transporting at least firstand second reactants through the first region, the first and secondreactants are maintained substantially together allowing reactants tointeract to form at least a first product in the first mixture;transporting the first mixture including the product to the secondregion wherein the product is separated from at least one of thereactants; and separating the product from at least one of thereactants.
 88. A method of performing integrated reaction and separationoperations in a microfluidic system, comprising: providing amicrofluidic device having at least first and second channel regionsdisposed therein, the first and second channel regions being connectedby a first connecting channel; introducing first reactants into thefirst channel region, the first reactants being contained within a firstmaterial region having a first ionic concentration, the first regionbeing bounded by second regions having a second ionic concentration, thesecond ionic concentration being lower than the first ionicconcentration; transporting the first and second material regions pastan intersection of the first channel region and the first connectingchannel, whereby at least a portion of the first material region isdiverted through the connecting channel and into the second channelregion.
 89. A method of performing integrated reaction and separationoperations in a microfluidic device, comprising: providing amicrofluidic device having a reaction channel portion and a separationchannel portion, the reaction channel portion being fluidly connectedand intersecting the separation channel portion at a first intersection;transporting at least a first reactant through the reaction channelportion within a first discrete fluid region, under conditions wherebythe reactant reacts to produce at least a first product, within thefirst fluid region, the first fluid region being bounded by at least asecond fluid region; detecting when the at least first fluid regionreaches the first intersection; injecting a portion of the at leastfirst fluid region into the separation channel; separating the productfrom the at least first reactant.
 90. The method of claim 89, whereinthe first fluid region has a conductivity higher than the second fluidregion, and the detecting step comprises detecting a change inconductivity in the first intersection when the first fluid regionreaches the first intersection.
 91. The method of claim 89, wherein atleast one of the first and second fluid regions comprises a markercompound, and the detecting step comprises detecting when the markercompound is present in the first intersection.
 92. A microfluidic devicefor performing integrated reaction and separation operations,comprising: a body structure having an integrated microscale channelnetwork disposed therein; a reaction region within the integratedmicroscale channel network, the reaction region having a mixture of atleast a first reactant and a first product disposed in and flowingthrough the reaction region, wherein the reaction region is configuredto maintain contact between the first reactant and the first productflowing therethrough; and a separation region in the integrated channelnetwork, the separation region in fluid communication with the reactionregion and being configured to separate the first reactant from thefirst product flowed therethrough.
 93. A microfluidic device foranalyzing electrokinetic mobility shifts of analytes, comprising: a bodystructure; a first microfluidic channel portion having substantially noelectrical field applied across its length; a second microfluidicchannel portion having an electrical field applied across its length,the second channel portion being fluidly connected to the first channelportion; and a pressure source in communication with at least one of thefirst channel portion and the second channel portion for moving amaterial through the first channel portion into the second channelportion.
 94. The microfluidic device of claim 93, comprising first andsecond electrodes in electrical contact at first and second ends of thesecond channel portion, respectively, each of the first and secondelectrodes being operably coupled to an electrical power source, forapplying the electric field across the length of the second channelportion.
 95. The microfluidic device of claim 93, wherein the pressuresource is a positive pressure source and is operably coupled to thefirst channel portion.
 96. The microfluidic device of claim 93, whereinthe pressure source comprises a negative pressure source, and isoperably coupled to the second channel portion, for drawing the analytesfrom the first channel portion into the second channel portion.
 97. Themicrofluidic device of claim 93, wherein the first channel portion isfluidly connected to a source of first and second analytes.
 98. Themicrofluidic device of claim 93, wherein the first channel portion isfluidly connected to a source of at least a third analyte.
 99. Themicrofluidic device of claim 98, further comprising a capillary elementextending out of the body structure, which capillary element includes acapillary channel disposed therein, the capillary channel being open ata first end and fluidly connected to the first channel portion at asecond end, and wherein fluid communication between the first channelportion and the source of at least a third analyte is provided bycontacting the open end of the capillary channel with a source of thethird analyte.
 100. The microfluidic device of claim 99, wherein thefirst and second electrodes are disposed in electrical contact withthird and fourth channels that are in fluid communication with thesecond channel portion at the first and second ends of the secondchannel portion, respectively.
 101. A method of analyzing an effect of afirst analyte on a second analyte, comprising: contacting the firstanalyte with the second analyte in a first microfluidic channel portionhaving substantially no electric field applied across its length;transporting at least a portion of the first analyte and second analyteto a second channel portion that is in fluid communication with thefirst channel portion and which has an electric field applied across itslength; measuring a change, if any, in an electrokinetic mobility of thesecond analyte in the second channel portion, a change in theelectrokinetic mobility of the second analyte being indicative of aneffect of the first analyte on the second analyte.
 102. The method ofclaim 101, wherein the effect of the first analyte on the second analyteis a binding of the first analyte to the second analyte, which resultsin a change of the electrokinetic mobility of the second analyte. 103.The method of claim 101, wherein the effect of the first analyte on thesecond analyte is a cleavage effect, which results is a change in anelectrokinetic mobility of the second analyte.
 104. The method of claim101, further comprising: contacting the first and second analytes in thefirst channel with a third analyte; and measuring a change in theelectrokinetic mobility of the second analyte in the presence of thethird analyte relative to a change in the electrokinetic mobility of thesecond analyte in the absence of the third analyte.
 105. The method ofclaim 101, wherein the second analyte has a detectable label associatedwith it.
 106. The method of claim 105, wherein the detectable labelcomprises an optically detectable label.
 107. The method of claim 106,wherein the optically detectable label comprises a fluorescent label.108. The method of claim 101, wherein the first ad second analytes aretransported into the second microfluidic channel portion by applying apressure differential between the first channel portion and the secondchannel portion
 109. A method of analyzing an electrokinetic mobilityshift in a first analyte, comprising: flowing the first analyte througha first microscale channel portion having substantially no electricalfield applied across it; introducing the first analyte into a secondmicrofluidic channel portion; applying an electric field across a lengthof the second microfluidic channel portion but not the firstmicrofluidic channel portion; measuring an electrokinetic mobility ofthe first analyte under the electric field applied in the second channelportion.
 110. The method of claim 109, wherein the first analytecomprises a product of an interaction between at least first and secondprecursor analytes, the first and second precursor analytes having afirst and second electrokinetic mobilities, respectively, and the firstanalyte having a third electrokinetic mobility.
 111. The method of claim110, wherein third electrokinetic mobility is different from at leastone of the first and second electrokinetic mobilities.
 112. The methodof claim 111, wherein the first precursor analyte comprises a detectablelabel, the detectable label becoming part of the first analyte when thefirst and second precursor analytes interact.
 113. The method of claim112, wherein the second electrokinetic mobility is different from thefirst electrokinetic mobility.
 114. The method of claim 113, wherein thefirst and second precursor analytes are moved from the first channelportion to the second channel portion by applying a pressuredifferential between the first and second channel portions to force thefirst and second precursor analytes into the second channel portion.115. Use of a microfluidic device for performing integrated reaction andseparation operations, the device comprising: a body structure having anintegrated microscale channel network disposed therein; a reactionregion within the integrated microscale channel network, the reactionregion having a mixture of at least first and second reactants disposedin and flowing through the reaction region, the mixture interacting toproduce one or more products, wherein the reaction region is configuredto maintain contact between the first and second reactants flowingtherethrough; and a separation region in the integrated channel network,the separation region in fluid communication with the reaction regionand being configured to separate the first reactant from the one or moreproducts flowed therethrough.
 116. Use of a microfluidic device forperforming integrated reaction and separation operations, the devicecomprising: a body structure having an integrated microscale channelnetwork disposed therein; a reaction region within the integratedmicroscale channel network, the reaction region having a mixture of atleast a first reactant and a first product disposed in and flowingthrough the reaction region, wherein the reaction region is configuredto maintain contact between the first reactant and the first productflowing therethrough; and, a separation region in the integrated channelnetwork, the separation region in fluid communication with the reactionregion and being configured to separate the first reactant from thefirst product flowed therethrough.
 117. Use of a device selected fromany one of the devices of claims 1-64 and 92-100 for practicing a methodselected from any one of the methods of claims 65-91 and 101-114. 118.An assay utilizing a use set forth in any one of claims 115-117.